hERG1 channel subunit composition mediates proton inhibition of rapid delayed rectifier potassium current (IKr) in cardiomyocytes derived from hiPSCs.

The voltage-gated channel, hERG1, conducts the rapid delayed rectifier potassium current (IKr) and is critical for human cardiac repolarization. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden death. At least two subunits form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG1a/1b abundance modulate IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue suggest that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation model to investigate how changes in relative hERG1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immatured hiPSC-CMs displays a 2-fold greater inhibition by extracellular acidosis (pH 6.3) compared with matured hiPSC-CMs. Quantitative RT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared with immatured hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by increased IKr. Silencing hERG1b's impact on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a N-terminal Per-Arnt-Sim domain reduced the magnitude of IKr proton inhibition in immatured hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and determines IKr proton sensitivity in hiPSC-CMs.

The N-linker region of hERG1a upregulates hERG1b potassium channels.

A major physiological role of hERG1 (human Ether-á-go-go-Related Gene 1) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the potassium current IKr in cardiomyocytes. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, long QT syndrome, and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood. Here, we report that the hERG1a N-terminal region expressed in trans with hERG1b markedly increased hERG1b currents and increased biotin-labeled hERG1b protein at the membrane surface. hERG1b channels with a deletion of the N-terminal 1b domain did not have a measurable increase in current or biotinylated protein when coexpressed with hERG1a N-terminal regions, indicating that the 1b domain was required for the increase in hERG1b. Using a biochemical pull-down interaction assay and a FRET hybridization experiment, we detected a direct interaction between the hERG1a N-terminal region and the hERG1b N-terminal region. Using engineered deletions and alanine mutagenesis, we identified a short span of amino acids at positions 216 to 220 within the hERG1a "N-linker" region that were necessary for the upregulation of hERG1b. We propose that direct structural interactions between the hERG1a N-linker region and the hERG1b 1b domain increase hERG1b at the plasma membrane. Mechanisms regulating hERG1a and hERG1b are likely critical for cardiac function, may be disrupted by long QT syndrome mutants, and serve as potential targets for therapeutics.

mTOR Modulation of IKr through hERG1b-Dependent Mechanisms in Lipotoxic Heart.

In the atria, the rapid delayed rectifier channel (IKr) is a critical contributor to repolarization. In lipotoxic atria, increased activity of the serine/threonine mammalian target of rapamycin (mTOR) may remodel IKr and predispose patients to arrhythmias. To investigate whether mTOR produced defects in IKr channel function (protein expression and gating mechanisms), electrophysiology and biochemical assays in HEK293 cells stably expressing hERG1a/1b, and adult guinea pig atrial myocytes were used. Feeding with the saturated fatty acid palmitic acid high-fat diet (HFD) was used to induce lipotoxicity. Lipotoxicity-challenged HEK293 cells displayed an increased density of hERG1a/1b currents due to a targeted and significant increase in hERG1b protein expression. Furthermore, lipotoxicity significantly slowed the hERG1a/1b inactivation kinetics, while the activation and deactivation remained essentially unchanged. mTOR complex 1 (mTORC1) inhibition with rapamycin (RAP) reversed the increase in hERG1a/1b density and inactivation. Compared to lipotoxic myocytes, RAP-treated cells displayed action potential durations (APDs) and IKr densities similar to those of controls. HFD feeding triggered arrhythmogenic changes (increased the IKr density and shortened the APD) in the atria, but this was not observed in low-fat-fed controls. The data are the first to show the modulation of IKr by mTORC1, possibly through the remodeling of hERG1b, in lipotoxic atrial myocytes. These results offer mechanistic insights with implications for targeted therapeutic options for the therapy of acquired supraventricular arrhythmias in obesity and associated pathologies.

Cotranslational association of mRNA encoding subunits of heteromeric ion channels.

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes.

hERG1a and hERG1b potassium channel subunits directly interact and preferentially form heteromeric channels.

Voltage-activated human ether-á-go-go-related gene (hERG) potassium channels are critical for the repolarization of cardiac action potentials and tune-spike frequency adaptation in neurons. Two isoforms of mammalian ERG1 channel subunits, ERG1a and ERG1b, are the principal subunits that conduct the IKr current in the heart and are also broadly expressed in the nervous system. However, there is little direct evidence that ERG1a and ERG1b form heteromeric channels. Here, using electrophysiology, biochemistry, and fluorescence approaches, we systematically tested for direct interactions between hERG1a and hERG1b subunits. We report 1) that hERG1a dominant-negative subunits suppress hERG1b currents (and vice versa), 2) that disulfide bonds form between single cysteine residues experimentally introduced into an extracellular loop of hERG1a and hERG1b subunits and produce hERG1a-hERG1b dimers, and 3) that hERG1a and hERG1b subunits tagged with fluorescent proteins that are FRET pairs exhibit robust energy transfer at the plasma membrane. Thus, multiple lines of evidence indicated a physical interaction between hERG1a and hERG1b, consistent with them forming heteromeric channels. Moreover, co-expression of variable ratios of hERG1a and hERG1b RNA yielded channels with deactivation kinetics that reached a plateau and were different from those of hERG1b channels, consistent with a preference of hERG1b subunits for hERG1a subunits. Cross-linking studies revealed that an equal input of hERG1a and hERG1b yields more hERG1a-hERG1a or hERG1a-hERG1b dimers than hERG1b-hERG1b dimers, also suggesting that hERG1b preferentially interacts with hERG1a. We conclude that hERG1b preferentially forms heteromeric ion channels with hERG1a at the plasma membrane.

Effects of cariprazine on hERG 1A and hERG 1A/3.1 potassium channels.

Cariprazine is a novel atypical antipsychotic drug that is widely used for the treatment of schizophrenia and bipolar mania/mixed disorder. We used the whole-cell patch-clamp technique to investigate the effects of cariprazine on hERG channels that are stably expressed in HEK cells. Cariprazine inhibited the hERG 1A and hERG 1A/3.1 tail currents at -50 mV in a concentration-dependent manner with IC50 values of 4.1 and 12.2 µM, respectively. The block of hERG 1A currents by cariprazine was voltage-dependent, and increased over a range of voltage for channel activation. Cariprazine shifted the steady-state inactivation curve of the hERG 1A currents in a hyperpolarizing direction and produced a use-dependent block. A fast application of cariprazine inhibited the hERG 1A currents elicited by a 5 s depolarizing pulse to +60 mV to fully inactivate the hERG 1A currents. During a repolarizing pulse wherein the hERG 1A current was deactivated slowly, cariprazine rapidly and reversibly blocked the open state of the hERG 1A current. However, cariprazine did not affect hERG 1A and hERG 1A/3.1 channel trafficking to the cell membrane. Our results indicated that cariprazine concentration-dependently inhibited hERG 1A and hERG 1A/3.1 currents by preferentially interacting with the open states of the hERG 1A channel, but not by the disruption of hERG 1A and hERG 1A/3.1 channel protein trafficking. Our study examined cariprazine's mechanism of action provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.

hERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms.

BACKGROUND: The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. OBJECTIVE: To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. METHODS: Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. RESULTS: The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. CONCLUSION: Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.